US Environmental Protection Agency Office of Pesticide Programs Office of Pesticide Programs Microbiology Laboratory Environmental Science Center, Ft. Meade, MD Standard Operating Procedure for Germicidal and Detergent Sanitizing Action of Disinfectants Test SOP Number: MB-27-00 Date Revised: 01-16-13 ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 1 of 19 SOP Number MB-27-00 Title Germicidal and Detergent Sanitizing Action of Disinfectants Test Scope This SOP describes the methodology used to determine the efficacy of food contact sanitizers against two organisms, Staphylococcus aureus and Escherichia coli. The methodology is based on AO AC method 960.09 (Germicidal and Detergent Sanitizing Action of Disinfectants - see reference 15.1), revision date 01/09/13 (pending publication by AOAC International). Application For product evaluations under the Antimicrobial Testing Program (ATP), a study protocol is developed which identifies the specific test conditions for a product sample such as contact time, dilutions, neutralizers, etc. Approval Date SOP Developer: Print Name: SOP Reviewer Print Name: Quality Assurance Unit Print Name: Branch Chief Print Name: Data SOP issued: Controlled copy number: Date SOP withdrawn: ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 2 of 19 TABLE OF CONTENTS Contents Page Number 1. DEFINITIONS 3 2. HEALTH AND SAFETY 3 3. PERSONNEL QUALIFICATIONS AND TRAINING 3 4. INSTRUMENT CALIBRATION 3 5. SAMPLE HANDLING AND STORAGE 3 6. QUALITY CONTROL 3 7. INTERFERENCES 3 8. NON-CONFORMING DATA 3 9. DATA MANAGEMENT 3 10. CAUTIONS 3 11. SPECIAL APPARATUS AND MATERIALS 4 12. PROCEDURE AND ANALYSIS 7 13. DATA ANALYSIS/CALCULATIONS 12 14. FORMS AND DATA SHEETS 12 15. REFERENCES 13 ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 3 of 19 1. Definitions Abbreviations/definitions are provided in the text. 2. Health and Safety Follow procedures specified in SOP MB-01, Laboratory Biosafety. The Study Director and/or lead analyst should consult the Material Safety Data Sheet for specific hazards associated with products. 3. Personnel Qualifications and Training Refer to SOP ADM-04, OPP Microbiology Laboratory Training. 4. Instrument Calibration Refer to SOP EQ-01, EQ-02, EQ-03, EQ-04 and EQ-05 for details on method and frequency of calibration. 5. Sample Handling and Storage Refer to SOP MB-22, Disinfectant Sample Preparation, and SOP COC-01, Chain of Custody Procedures. 6. Quality Control For quality control purposes, the required information is documented on the appropriate form(s) (see section 14). It is critical to maintain the highest standards of good laboratory practices and aseptic technique during all manipulations and handling of stock cultures. 7. Interferences 1. Contamination of stock and test cultures will negatively impact disinfectant efficacy testing. 2. Any level of contamination in treated or numbers control plates that interfere with reading the results will invalidate the test. 3. Prior to use visually inspect all agar plates and discard any plates with evidence of contamination. 4. For contamination following the incubation phase, if contamination is evident and interferes with the enumeration of the test organism, record as a contaminant(s). Data from only one dilution may be used to calculate the final CFU/plate. 8. Non- conforming Data 1. Sterility and/or viability controls do not yield expected results. 2. Management of non-conforming data will be specified in the study protocol; procedures will be consistent with SOP ADM-07, Non- Conformance Reports. 3. For a valid test numbers control counts must fall between 7.0 - 8.0 logs. 9. Data Management Data will be archived consistent with SOP ADM-03, Records and Archives. 10. Cautions 1. Verify the volume of dilution blanks, and neutralizer tubes in advance and adjust accordingly. 2. Media indicated in section 11.2.a and 11,2.b for rehydrating lyophilized cultures are specified in the ATCC Product Information Sheet. Upon purchase of new organisms, verify that media requirements have not ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 4 of 19 changed by checking the new ATCC Product Information Sheet. 3. Strict adherence to the protocol is necessary for the validity of the test results. Microbiological methods are technique-sensitive and technique- oriented; thus, exact adherence to the method, good laboratory practices, and quality control are required for proficiency and validity of the results 4. Do not allow the pipette to touch the neck or side of the flask during the addition of the culture suspension. 5. Plating should be completed within 1 hour after the initiation of serial dilutions. 6. For spread plating: ensure that the entire surface of the agar plate is dry before adding inoculum. If necessary, leave the agar plates uncovered in the biological safety cabinet (BSC) until the moisture has been completely absorbed into the medium. 7. Diluted product must be used within three hours of preparation unless otherwise specified. 8. Detergents used in washing glassware may leave residues which are bacteriostatic. Test for inhibitory residues on glassware periodically according to SOP QC-03, Glass Washing and Detergent Residues Test. 9. Sterility and performance of medium can be verified if the subculture medium is an adequate growth medium as stated in SOP MB-10, Media and Reagents Used in Microbiological Assays Including Performance Assessment and Sterility Verification. 11. Special Apparatus and Materials 1. Test organisms. Escherichia coli (ATCC No. 11229) and Staphylococcus aureus (ATCC No. 6538) obtained directly from a reputable supplier (e.g., ATCC). 2. Culture media (e.g., nutrient agar). Note: Commercial dehydrated media made to conform to the recipes provided in AOAC Method 960.09 may be substituted. a. Trypticase Soy Agar (TSA). Prepare according to manufacturer's instructions. Used for the generation of frozen stock cultures for S. aureus. b. Tryptic Soy Broth (TSB). Prepare according to manufacturer's instructions. Used for the generation of frozen stock cultures for S. aureus. c. Nutrient broth. Boil 5 g beef extract (paste or powder), 5 g NaCl, and 10 g peptone (anatone) in 1 L H2O for 20 minutes and dilute to volume with de-ionized water; adjust to pH 6.8 ± 0.1. Filter through paper (Whatman No. 4, or equivalent), place 10 mL portions in 20 x 150 mm test tubes, and steam sterilize 20 min at 121°C. Use this broth for the preparation of Nutrient agar plates. d. Nutrient agar (AOAC): Dissolve Bacto agar to 1.5% in nutrient broth and adjust to pH 1.2-1 A. Steam sterilize for 20 min at 121°C. ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 5 of 19 Dispense into plates. Media is used for the generation of frozen stock cultures for E. coli. e. Nutrient agar -A (NA-A): Boil 3 g beef extract, 5 g peptone and 15 g salt free agar in 1 L de-ionized water. Dot not use premixed dehydrated media. Dispense 10 mL portions in 20 x 150 mm tubes or 20 mL portions in 25 x 150 mm tubes and steam sterilize for 20 min at 121°C. Slant tubes after sterilization and let cool. Media is used for daily transfers of test cultures. f. Nutrient agar - B (NA-B): Boil 3 g beef extract, 5 g peptone and 30 g salt free agar in 1 L de-ionized water. Dot not use premixed dehydrated media. Steam sterilize for 20 min at 121 °C. Temper media prior to dispensing 20-30 mL portions into sterile Petri dishes. Media is used for development of the final test culture. 3. Subculture media: choose the appropriate recovery agar and neutralizer to inactivate the test chemical, for example: a. Trypone glucose extract agar plus Neutralizer (TGEA-N): Combine 24 g of dehydrated media with 975 mL de-ionized water and 25 mL stock neutralizer if necessary. Steam sterilize for 15 min at 121°C. Media used for the recovery of test organisms from treated samples. b. Tryptone glucose extract agar (TGEA): Prepare according to the manufacturer's instructions. Plating medium is used for enumeration of numbers control samples. 4. Cryoprotectant solution (TSB with 15% glycerol). Suspend 7.5 g tryptic soy broth in 212.5 mL de-ionized water. Add 37.5 g glycerol and stir until dissolved; boil to dissolve completely. Dispense into bottles and autoclave for 15 minutes at 121°C. Solution is used for the preservation of frozen stock cultures. 5. Neutralizer Stock Solution (NSS): Mix 40 g Lecithin, 280 mL polysorbate 80 and 1.25 mL 0.25 M phosphate buffer stock solution. Dilute with de- ionized water to 1 L and adjust pH to 7.2. Dispense in 100 mL portions and steam sterilize for 20 min at 121°C. 6. Neutralizer blanks (NB): For use with < 200ppm quaternary ammonium compounds. Mix 100 mL neutralizer stock solution, 25 mL 0.25 M phosphate buffer stock solution, and 1675 mL of de-ionized water. Dispense into appropriate size vessel and sterilize for 20 min at 121°C. Alternate neutralizers may be used as necessary. 7. Phosphate buffer stock solution (PBSS): 0.25M. Dissolve 34 g KH2PO4 in 500 mL de-ionized water in 1 L volumetric flask. Adjust pH to 7.2 with ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 6 of 19 1 N NaOH, and dilute to 1 L volume mark. Sterilize by filtration. 8. Phosphate buffer solution (PBS). Add 1.25 mL of phosphate buffer stock solution and 8.75 g of NaCl to a volumetric flask; fill with de-ionized water to the 1000 mL mark and mix. A pH of approximately 7.0 is desirable. Sterilize by filtration or autoclaving at 121°C for 15-20 min. Alternative PBS formulations with the same pH may be used (e.g., dilute commercially prepared 10x PBS solution to lx using de-ionized water). 9. Phosphate buffer dilution water stock solution (PBDW-SS). Dissolve 34.0 g of potassium dihydrogen phosphate (KH2PO4) in 500 mL de-ionized water. Adjust pH to 7.2 ± 0.2 with 0.1 N NaOH or 0.1 N HC1 and bring to 1000 mL with de-ionized water. Alternative phosphate buffers with the same pH may be used (e.g., commercially prepared 10X PBS solution). 10. Phosphate buffer dilution water (PBDW): Add 1.25 mL of 0.25 M phosphate buffer stock solution to 1 L de-ionized water. Dispense into appropriate size vessel and sterilize for 20 min at 121°C. Used for numbers control and dilution blanks. 11. Tween-80 (polysorbate 80). 12. PBS (lx) with 0.1% Tween 80 (PBS + T80): Add 100 mL PBS 10x solution and 1 mL Tween 80 to a volumetric flask; fill with de-ionized water to the 1000 mL mark and mix thoroughly. Sterilize by filtration. 13. Sterile water. Use reagent-grade water free of substances that interfere with analytical methods. Any method of preparation of reagent-grade water is acceptable provided that the requisite quality can be met. Reverse osmosis, distillation, and deionization in various combinations all can produce reagent-grade water when used in the proper arrangement. See Standard Methods for the Examination of Water and Wastewater and SOP QC-01, Quality Assurance of Purified Water for details on reagent- grade water. 14. Blood Agar (BAP). Commercially purchased plates. Media used for the presumptive identification of the test microbes. 15. Mannitol Salt Agar (MSA). Combine 111 g of dehydrated media with 1 L de-ionized water and mix thoroughly. Steam sterilize for 15 min at 121°C. Media used for the presumptive identification of the test microbes. 16. Xylose lysine deoxycholate agar (XLD). Commercially purchased plates. Media used for the presumptive identification of the test microbes. 17. Glassware. For sanitizer efficacy and numbers control, use 250 mL wide mouth Erlenmeyer flasks. For measuring sanitizers and dilution blanks ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 7 of 19 use 100 mL graduated cylinders. For glassware used to prepare test chemical, refer to SOP MB-22. 18. Petri dishes. Sterile plates, 20 mm x 100 mm in size. 19. Recirculating chiller unit. For maintaining specified temperature of the test chemical (capable of maintaining 25 ± 1°C). 20. Spectrophotometer. Calibrated. 21. Test tube racks. Any convenient style. 22. Transfer loops. Make 4 mm ID single loop at end of 50-75 mm (2-3 in.) Pt or Pt alloy wire No. 23 B&S gage or 4 mm loop fused on 75 mm (3 in.) shaft (available from Johnson Matthey, West Chester, PA 19380, USA). Fit other end in suitable holder. Bend loop at 30° angle with stem. Commercially available 4 mm ID transfer loops may also be used. Volumetric transfer devices may be used instead of transfer loops (e.g., micro volume pipette). 23. Timer. For managing timed activities, any certified timer that can display time in seconds. 24. Micropipettes. For performing culture transfers and serial dilutions. 25. Whatman No. 2 filter paper. Sterile. 26. Gram stain kit. 27. Indole reagent. For the identification of E. coli. 28. Catalase Reagent. For the identification of S. aureus. 29. Staphaurex Reagent Kit. For the identification of S. aureus. 12. Procedure and Analysis Prior to testing, perform the neutralization assay to determine if the prescribed neutralizer is appropriate for the test chemical. 12.1 Test Culture Preparation Refer to Attachment 2. A for preparation of the frozen stock cultures. Refer to MB-02: Tracking of Test Microorganisms, Section 12 for the tracking and transfer notations for the test microbes. Reinitiate new frozen stock cultures every 18 months with a new lyophilized culture. a. Defrost a single cryovial of frozen stock culture at room temperature and briefly vortex to mix. Streak one loopful of the thawed frozen stock onto a NA - A slant and incubate at 36 ± 1°C for 24 ± 2 h. Only one daily transfer is required prior to the initiation of the final ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 8 of 19 test culture. b. For the final test culture, add 5 mL of PBDW to a NA - A slant (daily culture). Using a sterile loop, dislodge growth from agar surface. Collect mixture and transfer to a flask containing 99 mL of PBDW. Mix throughly. Add 200 |iL of the mixture to inoculate a minimum of 5 NA - B plates and to create a bacterial lawn. Incubate at 36 ± 1°C for 24 ± 2 h. c. Record all culture transfers on the Organism Culture Tracking Form (see section 14). 12.2 Test Culture Harvesting a. After incubation, add a minimum of 5 mL of PBS + Tween 80 to each plate. Using a sterile rod, gently dislodge culture from agar surface, avoid disrupting agar. Combine culture from all plates and mix thoroughly. b. Filter culture through sterile Whatman No. 2 filter paper using a vacuum source; collect filtered culture into a sterile vessel. c. Standardize the test culture, if necessary using PBDW to achieve a final test culture microbe population of 1.0 x 109 CFU/ mL and 1.0 x 1010 CFU/mL (9-10 logio/mL). 12.3 Test chemical Sample Preparation a. Prepare test chemical sample per SOP MB-22. 1) Ready-to-use products are tested as received; no dilution is required. 2) If hard water is required as the diluent, prepare synthetic hard water as described in SOP MB-22. b. Equilibrate water bath and allow it to come to 25 ± 1°C or the temperature specified (±1°C). Prepare the test chemical dilutions within 3 h of performing the assay. c. Dispense 99 mL aliquots of the diluted test chemical or ready-to-use product into sterile wide mouth Erlenmeyer flasks. Prepare one flask per test microbe, for each germicide to be tested. d. Place flasks in the equilibrated water bath for approximately 10 min to allow test solution to come to specified temperature. e. Record the temperature of the water bath and recirculating chiller before and after testing on the Germicidal and Detergent Sanitizing Action of Disinfectants Method Test Information and Culture Preparation Sheet (see section 14). f. In addition, prepare a similar flask containing 99 mL of PBDW to use for numbers control for each organism. ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 9 of 19 12.4 Test Procedure a. Add 1 mL of test culture to the test flask as follows: b. Whirl flask, stopping just before suspension is added, creating enough residual motion of liquid to prevent pooling of suspension at the point of contact with test sample. c. Add suspension midway between center and the inner edge of the flask with tip of pipette slightly immersed in test solution. Avoid touching the neck or side of flask during addition. Swirl flask to thoroughly mix contents. NOTE: The flasks containing test culture x test chemical are referred to as the treated samples. d. At 30 ± 3 seconds after addition of the test culture, transfer a 1 mL aliquot from the test flask (test culture x test chemical) to a tube containing 9 mL neutralizer blank and mix well. This corresponds to 10"1 dilution tube. Record timed events on the Germicidal and detergent Sanitizing Action of Disinfectants Method Time Recording Sheet for Transfers (see section 14). e. Treated samples plating.— From 10"1 tube (i.e., 9 mL neutralizer tube inoculated with 1 mL of exposed culture)., plate four 1 mL aliquots and four 0.1 mL aliquots onto TGEA - N plates, for a total 1 2 of 8 plates per treated sample. This will result in 10" and 10" dilutions respectively. Incubate plates at 36±1°C for 24-30 hours. f. Following incubation, count colonies on TGEA-N plates. Counts over 300 are recorded as TNTC. Record plate counts on the Germicidal and Detergent Sanitizing Action of Disinfectants Method Results Sheet (see section 14). 12.5 Numbers Control a. Numbers control assay should be conducted within 5 minutes of completion of treated samples. b. In a sterile 250 mL wide mouth Erlenmeyer flask containing 99 mL sterile PBDW, add 1 mL of the test culture (same test culture used for the treated samples) as follows: c. Whirl flask, stopping just before suspension is added, creating enough residual motion of liquid to prevent pooling of suspension at the point of contact with test sample. d. Add suspension midway between center and the inner edge of the flask with tip of pipette slightly immersed in test solution. Avoid touching to the neck or side of flask during addition. Swirl flask to thoroughly mix contents. e. Numbers control plating.— Within 30 seconds of addition of test culture, transfer 1 mL aliquot from the test flask (test culture x PBDW) into a tube containing 9 mL of neutralizer and mix well. This corresponds to 10"1 dilution tube. Make serial 10-fold dilutions in 9 mL PBDW, out to 10"6. ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 10 of 19 f. Plate four 1 mL aliquots and four 0.1 mL aliquots from the 10"6 dilution tube onto TGEA plates, for a total of 8 plates per numbers control sample. This will result in 10"6 and 10"7 dilutions respectively. g. Incubate plates at 36±1°C for 24-30 hours. h. Following incubation, count colonies on TGEA plates. Counts over 300 are recorded as TNTC. Record plate counts on the Germicidal and Detergent Sanitizing Action of Disinfectants Method Results Sheet (see section 14). i. For a valid test, the numbers control must fall between 7.0 - 8.0 logio/mL. 12.6 Sterility controls a. Neutralizer blank - plate 1 mL from a previously unopened tube onto a TGEA plate. b. Sanitizer - Plate 1 mL of the product onto a TGEA plate. c. Diluent - Plate 1 mL of the diluent onto a TGEA plate. d. PBDW - Plate 1 mL of the diluent onto a TGEA plate. e. Incubate all plates at 36±1°C for 24 -30 hours, record results. f. To be considered valid, no growth should be observed on any of the sterility controls. 12.7 Neutralization Confirmation Test a. A neutralization confirmation test should be performed prior to or concurrently with the sanitizer evaluation assay. The neutralization assay must demonstrate the recovery of a low level test organism population (e.g., 10-100 CFU/mL) in the recovery media (i.e. TGEA-N and TGEA). b. Test culture Titer (TCT) .— Add 0.1 mL of the test organism, which has been serially diluted to target between 10-100 CFU/mL to 10 mL of PBDW and mix thoroughly. Dilutions 10"4 and 10"5 should provide the range of 10-100 CFU/mL. Hold the mixture for a minimum of two minutes. Plate 0.1 mL aliquots in duplicate onto TGEA. Incubate plates at 36±1°C for 24-30 hours and record number of colonies. c. Neutralization Confirmation Treatment (NCT).— Add 1 mL of the test chemical to 9 mL of the prescribed neutralizer and mix thoroughly. Within 30 seconds, inoculate the sample with 0.1 mL of the test organism used for the TCT. Mix thoroughly. Hold the mixture for a minimum of two minutes. Plate 0.1 mL aliquots in duplicate onto TGEA-N. Incubate plates at 36±1°C for 24-30 hours and record number of colonies. d. Neutralization Toxicity Treatment (NTT) .— Add 0.1 mL of the test ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 11 of 19 organism used for the TCT to 10 mL of the prescribed neutralizer and mix thoroughly. Hold the mixture for a minimum of two minutes. Plate 0.1 mL aliquots in duplicate onto TGEA-N. Incubate plates at 36±1°C for 24-30 hours and record number of colonies. e. Plates that have colony counts over 300 are reported as TNTC. Record the counts on the Neutralization Confirmation Assay Results Sheet (see section 14). f. Neutralization Results and Calculations: In order to demonstrate effective neutralization of the sanitizer, differences between treatments should not exceed 1.0 log (e.g., TCT minus NCT). To calculate CFU/mL for Neutralization Confirmation Test use the following equation.— CFU/mL = (avg. CFU for 10~x ) + (avg. CFU for 10~y ) 10 x and 10 v where 10 x and 10 v are the dilutions plated. Two plates per dilution are plated for all treatments. Alternatively if only one dilution is plated use the same equation for only one dilution. Use counts of 0 to 300 for calculation purposes. Score counts >300 as TNTC (too numerous to count). NOTE: A spreadsheet will be used for data analysis and calculations associated with the neutralization confirmation assay. 12.8 Results For the test chemical to be considered efficacious, a > 5 log reduction is required with a contact time of 30 seconds. Retestingguidance: For tests where the product meets the performance standard and the numbers control mean logio density value is above 8.0, no retesting is necessary. For tests where the product fails to meet the performance standard and the numbers control mean lotio density is below 7.0, no retesting is necessary. 12.9 Confirmatory Steps for Test Microbes a. Presumptive identification of the test microbes will be conducted when results are indicative of a failing efficacy evaluation or when results are inconclusive or have discrepancies. b. Representative growth from one plate per treatment sample should be confirmed by Gram stain and growth characteristics on general and selective media. c. Gram stains are performed on smears taken from the treatment sample plates. For the additional confirmatory tests, a smear of culture from each selected treatment sample plate is streaked onto BAP and selective media appropriate for the test organism and incubated for 18-24 hours at 36 ± 1°C. See Attachment 1 for Gram ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 12 of 19 stain reactions, cell morphology, and colony characteristics on solid media. d. If characteristics on general and/or selective media are unusual, then further confirmation will be conducted by using the following: rapid indole test for E. coli and Catalase and Staphaurex test for S. aureus. Refer to the appropriate Confirmation Flow Chart for S. aureus, and E. coli (see Attachment 3). e. If confirmatory testing determines that the identity of the organism was not the test organism the entry on the results sheet must be annotated to indicate a contaminant was present. 13. Data Analysis/ Calculations Calculations will be computed using a Microsoft Excel spreadsheet (see section 14). Both electronic and hard copies of the spreadsheet will be retained. To calculate CFU/mL, use the following equation.— CFU/mL = (avg. CFU for 10~x ) + (avg. CFU for 10~y ) 10 x and 10 v where 10 x and 10 v are the dilutions plated. Four plates per dilution are plated for treated samples and numbers control samples. Use counts of 0 to 300 for calculation purposes. Score counts >300 as TNTC (too numerous to count). a. Calculate the mean logio density (LD) for number control plates b. Calculate the mean logio density (LD) for treated samples plates. c. Calculate the logio reduction (LR) for treated samples: Logio reduction = mean logio numbers control - mean logio treated sample 14. Forms and Data Sheets 1. Attachment 1: Typical Growth Characteristics of strains of S. aureus and E. coli 2. Attachment 2: Culture Initiation Flow Chart for S. aureus and E. coli 3. Attachment 3: Confirmation Flow Charts for S. aureus and E. coli 4. Test Sheets. Test sheets are stored separately from the SOP under the following file names: Organism Culture Tracking Form MB-27-00 Fl.docx Test Microbe Confirmation Sheet (Quality MB-27-00 F2.docx Control) Germicidal and Detergent Sanitizing Action of MB-027-00 F3.docx Disinfectants Method: Test Information and Culture Preparation Sheet ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 13 of 19 Germicidal and Detergent Sanitizing Action of MB-27-00 F4.docx Disinfectants Method: Serial Dilution/Plating Tracking Form Germicidal and Detergent Sanitizing Action of MB-27-00 F5.docx Disinfectants Method: Titer of Final Test culture Form Germicidal and Detergent Sanitizing Action of MB-27-00 F6.docx Disinfectants Method: Results Sheet Germicidal and Detergent Sanitizing Action of MB-027-00 F7.docx Disinfectants Method: Time Recording Sheet for Transfers Germicidal and Detergent Sanitizing Action of MB-27-00 F8.docx Disinfectants Method: Test Microbe Confirmation Sheet Germicidal and Detergent Sanitizing Action of MB-27-00 F9.docx Disinfectants Method: Neutralization Confirmation Assay - Neutralization Confirmation Control Germicidal and Detergent Sanitizing Action of MB-27-00 FlO.docx Disinfectants Method: Neutralization Toxicity Control Germicidal and Detergent Sanitizing Action of MB-27-00 F1 l.docx Disinfectants Method: Neutralization Confirmation Assay - Test culture Control Germicidal and Detergent Sanitizing Action of MB-27-00 F12.docx Disinfectants Method: Neutralization Confirmation Assay Results Sheet 15. References 1. Official Methods of Analysis. 2013. 18th Ed., AOAC INTERNATIONAL, Gaithersburg, MD. Method 960.09: Germicidal and Detergent Sanitizing Action of Disinfectants. Revised First Action 2013. Pending publication by AOAC International. 2. Package Insert - Gram Stain Kit and Reagents. Becton, Dickinson and Company. Part no. 882020191JAA. Revision 07/2011. 3. Package Insert - Catalase Reagent Droppers. Becton, Dickinson and Company. Part no. L001237. Revision 06/2010. 4. Package Insert - Staphaurex Plus*. Remel. Part no. R30950102. Revised 11/23/07. ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 14 of 19 5. Package Insert - Rapid Indole Reagent Droppers. Becton, Dickinson and Company. Part no. L001237. Revision 06/2010. ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 15 of 19 Attachment 1 Typical Growth Characteristics of strains of P. aeruginosa and is. coli (see ref. xxx). S. aureus* E. coli* Gram slain reaction (+) (-) Typical Growth Characteristics on Solid Media BAP small, circular, yellow or white, glistening, beta hemolytic large, round, white colonics, non- hemolytic. Mannitol Salt circular, small, yellow colonics, agar turning riuorcsccnl yellow No Growth XLD agar No growth large, flat, yellow colonics with no black center. L-EMB Agar No Growth large colonics, circular. Blue-black with green metallic sheen Typical Microscopic Characteristics Cell dimensions 0.5-1.5 |im in diameter 0.5 x 1-3 |im in diameter Cell appearance spherical, occurring singly, in pairs and tetrads, sometimes forming irregular clusters varying from almost coccoid forms to long rods, occurring singly, in pairs and in short chairs. Motile or non- motile, if motile with flagella. *After 24±2 hours *After 24±2 hours ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 16 of 19 Attachment 2 Culture Initiation and Stock Culture Generation Flow Chart for S. aureus and E. coli. ® Rehydrate ampule. i © Transfer entire rehydrated pellet to TUBE A. Ampule Incubate TSBor NB TUBE A TUBE A (pre-incubation) (post-incubation) ©Stock Culture Generation Inoculate TSA orNA plates with 100 ^iL culture from TUBE A; incubate. Harvest inoculum from plates. © Culture ID & Quality Control Prepare frozen stock cultures BAP /\ Selective media Gram Additional Stain confirmation steps (see Attachment 3) Al. Preparation of Frozen Stock Cultures. Refer to SOP MB-02 for establishment of the organism control number. a. Initiate new stock cultures from lyophilized cultures of Staphylococcus aureus (ATCC 6538), and Escherichia coli (ATCC 11229) from ATCC within 18 months. b. Open ampule of freeze dried organism as indicated by ATCC. Using a tube containing 5-6 mL of TSB for S. aureus or NB for E. coli, aseptically withdraw 0.5 to 1.0 mL and rehydrate the lyophilized culture. Aseptically transfer the entire rehydrated pellet back into the original tube of broth designated as "TUBE A". Mix well. c. Incubate broth culture (TUBE A) at 36 ± 1°C for 24 ± 2 hours. Record all manipulations on the Organism Culture Tracking Form (see section 14). ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 17 of 19 d. Using a sterile spreader, inoculate a sufficient number of TSA plates for S. aureus or NA for E. coh, (e.g., 5 to 10 plates per organism) with 100 |iL each of the culture to create a bacterial lawn. Incubate plates at 36 ± 1°C for 24 ± 2 h. e. Following incubation, add 5 mL cryoprotectant solution (TSB with 15% v/v glycerol) to the surface of each agar plate. Re-suspend the cells in this solution using a sterile spreader or a sterile swab and aspirate the cell suspension from the surface of the agar. Transfer the suspension into a sterile vessel. f. Mix the pooled contents of the vessel thoroughly. Immediately after mixing, dispense approximately 1.0 mL aliquots into cryovials (e.g., 1.5 mL cyrovials). Perform QC of stock cultures concurrently with freezing (see section A2: QC of Stock Cultures). g. Store the cryovials at -70°C or below; these are the frozen stock cultures. Store stock cultures up to 18 months. These vials are single-use only. A2. QC of Stock Cultures. a. Conduct QC of the pooled culture concurrently with freezing. Streak a loopful on a plate of BAP. In addition, for S. aureus streak a loopful onto selective media (i.e., MSA); for E. coli, streak a loopful onto XLD. Incubate all plates at 36 ± 1°C for 24 ± 2 hours. b. Following the incubation period, record the colony morphology as observed on the BAPs and selective media plates (including the absence of growth) and Gram stain. See Attachment 1 for details on cell and colony morphology, colony characteristics on selective media, and stain reactions. c. For each organism, perform a Gram stain from growth taken from the BAPs according to the manufacturer's instructions. Observe the Gram reaction by using brightfield microscopy at 1000X magnification (oil immersion). d. For additional confirmation steps refer to the appropriate Confirmation Flow Chart for S. aureus and E. coli (see Attachment 3). Refer to 15.3-15.5 for instructions. e. Record all confirmation results on the Test Microbe Confirmation Sheet (Quality Control) (see section 14). ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 18 of 19 Attachment 3 Confirmation Flow Chart for S. aureus S. aureus Identification Identified as S1. aureus ------- SOP No. MB-27-00 Date Revised 01-16-13 Page 19 of 19 Attachment 3 (cont.) Confirmation Flow Chart for is. coli E. coli Identification Take one colony from sample plate onto BAP and XLD. Confirm GNR 1 BAP - Round colonies, non -hemolytic XLD - Yellow colonies. No black center. Perform Spot Indole test from BAP colony (Positive result = pink coloration in area with culture) Not E. coli Negative Positive E. coli ------- |